Establishment of HIV Latency in Primary CD4 Cells Is due to

JOURNAL OF VIROLOGY, July 2010, p. 6425–6437 Vol. 84, No. 130022-538X/10/$12.00 doi:10.1128/JVI.01519-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Establishment of HIV Latency in Primary CD4� Cells Is due toEpigenetic Transcriptional Silencing and P-TEFb Restriction�

Mudit Tyagi, Richard John Pearson,† and Jonathan Karn*Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106

Received 21 July 2009/Accepted 15 April 2010

The development of suitable experimental systems for studying HIV latency in primary cells that permitdetailed biochemical analyses and the screening of drugs is a critical step in the effort to develop viraleradication strategies. Primary CD4� T cells were isolated from peripheral blood and amplified by antibodiesto the T-cell receptor (TCR). The cells were then infected by lentiviral vectors carrying fluorescent reportersand either the wild-type Tat gene or the attenuated H13L Tat gene. After sorting for the positive cells andreamplification, the infected cells were allowed to spontaneously enter latency by long-term cultivation on theH80 feeder cell line in the absence of TCR stimulation. By 6 weeks almost all of the cells lost fluorescent proteinmarker expression; however, more than 95% of these latently infected cells could be reactivated after stimu-lation of the TCR by �-CD3/CD28 antibodies. Chromatin immunoprecipitation assays showed that, analo-gously to Jurkat T cells, latent proviruses in primary CD4� T cells are enriched in heterochromatic markers,including high levels of CBF-1, histone deacetylases, and methylated histones. Upon TCR activation, there wasrecruitment of NF-�B to the promoter and conversion of heterochromatin structures present on the latentprovirus to active euchromatin structures containing acetylated histones. Surprisingly, latently infected pri-mary cells cannot be induced by tumor necrosis factor alpha because of a restriction in P-TEFb levels, whichcan be overcome by activation of the TCR. Thus, a combination of restrictive chromatin structures at the HIVlong terminal repeat and limiting P-TEFb levels contribute to transcriptional silencing leading to latency inprimary CD4� T cells.

The introduction of highly active antiretroviral therapy(HAART) in the mid 1990s led to a dramatic increase inpatient longevity due to the ability of antiretroviral drugs tosuppress HIV replication to below threshold detection levels(�50 copies HIV RNA/ml) (23, 52). Unfortunately, despitethe intensive therapy, there is continuing viral replication atlevels below the limits of detection of most clinical assays dueto inefficient antiviral pharmacodynamics that create environ-ments where drug potency is reduced (12, 13, 41, 43). Forexample, there is recent evidence for ongoing HIV replicationin gut-associated lymphoid tissue during long-term antiretro-viral therapy (7). A second cause of HIV treatment failure isthe creation of a subpopulation of HIV-infected CD4� T lym-phocytes that harbors latent replication-competent proviruses.Since no viral proteins are produced, the latently infected cellscannot be recognized by the antiviral immune response and arehighly resistant to antiretroviral therapy. The development ofthese latent and slowly replicating viral reservoirs during HIVinfections has immense practical consequences for treatmentof HIV infections because it provides a mechanism that allowsthe virus to evade immune clearance and the effects of antiviraldrugs while still retaining an ability to quickly revert to theproductive state upon interruption of drug therapy or in re-sponse to cellular activation signals (6, 17).

Multiple complementary mechanisms are required to silenceHIV transcription and permit its entry into latency. AlthoughHIV silencing can readily occur in transformed cell lines, sev-eral features of the metabolism of resting CD4 cells ensure thatlatent proviruses remain transcriptionally inactive for long pe-riods. First, a key factor contributing to the restricted tran-scriptional initiation that is characteristic of HIV transcrip-tional silencing is the sequestration of the cellular initiationfactors NF-�B and NFAT in the cytoplasm of quiescent T cells(28, 37). The second major transcriptional block seen in la-tently infected cells is the incorporation of the P-TEFb elon-gation factor into an inactive complex containing HEXIM and7SK RNA (8, 56). This restricts P-TEFb levels in the cell andcreates a block to efficient transcription elongation from theHIV promoter. In addition, posttranscriptional restrictionsfurther reduce HIV gene expression. For example, limitingnuclear levels of the PTB splicing factor in quiescent cells leadsto a block to the export of HIV-specific RNA transcripts (32).Finally, miRNAs that inhibit translation of HIV mRNAs mayalso play an important role in maintaining HIV latency(24, 38).

Entry into latency is also strongly correlated with the recruit-ment of histone deacetylases (HDACs) to the HIV long ter-minal repeat (LTR) (9, 50). For example, we have recentlydemonstrated that CBF-1 (for latency C-promoter binding fac-tor 1), a DNA-binding protein that plays a central role in theNotch signaling pathway, can direct transcriptional silencing ofthe HIV LTR through recruitment of HDAC-1 (49). TheHDACs help to establish restrictive chromatin structures thatlimit HIV transcriptional initiation and elongation. Additionalchromatin restrictions due to histone H3 methylation by his-tone methyltransferase Suv39H1, lead to the accumulation of

* Corresponding author. Mailing address: Department of MolecularBiology and Microbiology, Case Western Reserve University, 10900Euclid Ave., Room W200, Cleveland, OH 44106-4960. Phone: (216)368-3915. Fax: (216) 368-3055. E-mail: [email protected].

† Present address: Department of Microbiology and Immunology,Stanford University School of Medicine, Stanford, CA 94305-5107.

� Published ahead of print on 21 April 2010.

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HP1 proteins on transcriptionally inactive proviruses (14, 35).We have also been able to monitor the progressive HIV si-lencing in isolated Jurkat T-cell clones and showed that latencyis associated with these chromatin modifications (40).

Although all silenced HIV proviruses appear to acquire re-strictive chromatin structures near the viral promoter, the cel-lular integration site can have a profound influence on theextent of proviral silencing. Viral integration into actively tran-scribed host genes can led to transcriptional interferencecaused by the elongating RNA polymerase II (RNAPII) tran-scribing through the viral promoter (15, 20, 33). Similarly,integration into heterochromatic regions can accelerate provi-ral silencing (25, 34).

Whereas there is compelling evidence that silencing throughhistone remodeling is a key feature mediating the establish-ment of HIV latency, the involvement of DNA methylation ismore controversial. Recent studies have shown that provirusesthat are poorly responsive to T-cell activation signals also tendto be hypermethylated (26). However, in many silenced HIVclones proviral expression does not correlate with DNA meth-ylation (42). Thus, it seems likely that although DNA methyl-ation is a powerful silencing mechanism for retroviruses, in thenatural setting, DNA methylation is not inevitably imposedand partially methylated promoters can still be reactivated(26).

Molecular studies of HIV latency have been severely ham-pered by the absence of reliable cellular models. The rarity oflatently infected cells in patients (less than 1 in 106 restingCD4� T cells in the peripheral circulation) makes it almostimpossible to isolate them in sufficient numbers for biochem-ical studies (41). As a result, virtually all molecular investiga-tions of HIV latency have involved the use of transformed celllines, such as the popular Jurkat T-cell line which carries afunctional T-cell receptor (TCR) signaling apparatus (25, 30,40). However, because the quiescent phenotype of the latentlyinfected CD4� T cells found in vivo is drastically different fromthe replicating and constitutively activated Jurkat T cells, manylaboratories are working to develop more suitable experimen-tal models for HIV latency using primary cells. A particularlyinformative model system has been developed by Zack andcoworkers, who have effectively used the HIV SCID-hu (Thy/Liv) mouse model to recapitulate the generation of latentlyinfected naive T cells during thymopoiesis both in vivo (4) andin vitro (5). Similarly, Cloyd and coworkers have reported thatlatently infected, quiescent CD4� T cells can be obtained bycultivating HIV-infected, activated normal CD4� T lympho-cytes on feeder cell layers (45).

In a significant recent set of advances, sophisticated cellculturing techniques have been used to recapitulate T-cell dif-ferentiation events in vitro and generate latently infected cells.First, Bosque and coworkers (26) have taken advantage ofpolarizing conditions to force activated T cells to differentiateand enter quiescence. Similarly, Marini et al. (36) used lowdoses of interleukin-7 (IL-7) to generate and maintain latentlyinfected memory CD4� T cells in vitro. Although promising,both methods are of limited use for biochemical analyses oflatent proviruses because the yields of viable latently infectedcells are low. Greater quantities of latently infected cells haverecently been obtained by Yang et al. (53), who used Bcl2 topartially transform primary CD4� T cells isolated from periph-

eral blood mononuclear cells (PBMC). We report here a novelex vivo method that permits, for the first time, the generationof large populations of latently infected primary CD4� T cells.We used this system to demonstrate that both creation ofheterochromatic structures on the HIV provirus and restric-tions in P-TEFb levels contribute to the establishment of HIVlatency in primary cells.

MATERIALS AND METHODS

Cell culture and lentiviral vectors. CD4� primary T cells and H80 cells weremaintained in RPMI 1640 medium supplemented with 10% fetal calf serum withor without IL-2 (20 U of recombinant human IL-2 [R&D Systems, Inc.]/ml). Thelentiviral vectors pHR�P-PNL-mCherry or pHR�P-PNL-d2EGFP vectors wereconstructed and pseudotyped HIV particles were packaged as described previ-ously (49).

Isolation, stimulation, and culture of CD4� T lymphocytes. The CD4� cellswere either isolated from the blood drawn from healthy donors or were isolatedfrom discarded tonsils. CD4� T cells were purified by negative selection methodusing a MACS kit (Miltenyi Biotechnology, Auburn, CA). Purified CD4� T cells(�98% pure) were stimulated for 4 days in RPMI 1640–10% fetal bovine serumwith 25 �l of �-CD3/CD28 antibodies conjugated to magnetic beads (DynalBiotech) per million cells, along with 20 U of IL-2/ml. One million cells wereinfected with VSV-G-pseudotyped HIV viruses. After 2 days, the fluorescentcells were purified by fluorescence-activated cell sorting (FACS) and again prop-agated in the presence of �-CD3/CD28 antibody-conjugated Dynal beads (25�l/106 cells) and 20 U of IL-2/ml for 2 to 3 weeks. Fresh medium was added every4 days, and the cultures were maintained at a density of 1.5 � 106 to 2.0 � 106

cells/ml. Once 0.5 � 108 to 1 � 108 cells were obtained they were placed on 30to 40% confluent H80 cell layers (45). Every 2 to 3 days, half of the culturemedium was replaced by fresh IL-2-containing medium, and every 2 weeks the Tlymphocytes were transferred to the fresh flasks of H80 feeder cells.

Flow cytometry. Cells were analyzed for fluorescent reporter gene expressionFACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Multi-color flow analysis of cell surface marker expression was performed by using aLSRII flow cytometer. Antibodies were conjugated to the fluorophores indicatedin the figure legends. Between 20,000 and 80,000 events were acquired for eachantibody and its appropriate isotype control. The data was analyzed with theFlowJo program.

Staining of cells with BrdU and Ki67. Cells were labeled for 18 h withbromodeoxyuridine (BrdU) using a commercial BrdU incorporation assay pro-tocol (BD Pharmingen). Approximately 3 � 105 labeled cells were permeabilizedusing 500 �l of Cytoperm (BD Biosciences) for 10 min in the dark at 37°C. Aftera washing step, the cells were incubated for 30 to 60 min with antibodies to BrdUand �-Ki67 or control antibodies. The unbound antibodies were then removed bywashing, and the cells were fixed and resuspended in 100 �l of 1% paraformal-dehyde in phosphate-buffered saline plus 200 �l of wash buffer prior to FACSanalysis.

ChIP assays. Chromatin immunoprecipitation (ChIP) was performed as pre-viously described (49). To activate cells, we used either 10 ng of tumor necrosisfactor alpha (TNF-�)/ml or 25 �l of �-CD3/CD28 antibodies bound to Dynalbeads (Dynal Biotech) per 106 cells. Most antibodies were purchased from SantaCruz, including anti-RNAPII (N-20), CBF-1(H-50), CIR (C-19), mSIN3A (AK-11), HDAC-1 (H-51), HDAC-2 (H-54), and p65 (C-20). Anti-acetylated his-tone-3 and histone-4 antibodies were obtained from Upstate.

Western blot analysis. Western blotting was performed according to standardprotocols. Anti-NF-�B, anti-CycT1, and anti-CDK-9 antibodies were obtainedfrom Santa-Cruz. Secondary horseradish peroxidase-conjugated anti-rabbit oranti-mouse antibodies were from Dako.

RESULTS

Efficient generation of pure populations of latently infectedCD4� memory T cells. We developed a novel ex vivo modelsystem to study HIV latency in primary CD4� T cells. Asoutlined in Fig. 1, pure populations (97%) of primary CD4�

T cells were isolated from PBMC by using a negative selectionMACS kit from Miltenyi Biotech. The cells were then ex-panded using �-CD3/CD28 antibody-coated beads in the pres-

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ence of IL-2. After 4 days, the cells were infected with HIV-derived vectors (Fig. 2A) that express fluorescent proteinreporter genes (either the short-lived d2EGFP or mCherry) inplace of the nef gene, as previously described (27, 49). LikeHIV itself, the viruses included the regulatory proteins Tat andRev, which provide a positive feedback circuit that enhancesHIV transcriptional elongation and export of mRNA from thenucleus. In the majority of our experiments, in order to in-crease the frequency of latently infected cells in the popula-tion, we utilized Tat carrying the H13L mutation. This partiallyattenuated Tat variant was originally identified in the U1 la-tently infected cell line (16, 44) and was earlier shown by us toexpedite HIV entry into latency (40). The use of an attenuatedTat mutant is consistent with recent studies which have shownthat latently infected cells accumulate Tat variants with re-duced transactivation potential (55). However, as describedbelow, latently infected cells can also be readily generated byusing wild-type Tat.

To obtain a homogeneous population of HIV-infected cells,cells expressing the fluorescent reporters were purified by cellsorting. The purified cells were further expanded with mag-netic beads with �-CD3/CD28 antibodies. After 4 to 6 weeks,the cell population reached between 50 � 106 and 100 � 106

cells; the magnetic beads were removed, and the cells wereplaced on H80 feeder cells in the presence of IL-2, as originallydescribed by Cloyd and coworkers (45).

As shown in Fig. 2B, cultivation of primary T cells infectedwith viruses carrying the H13L mutation in Tat on the feederlayers led to the progressive loss of HIV gene expression andthe entry of the cells into a largely quiescent state character-ized by a dramatic reduction in cell size. After 6 weeks, 92.13%of the cells lost virtually all d2EGFP reporter gene expression(Fig. 2B). As shown in Fig. 2B and 8B, comparison of thed2EGFP profiles in the silenced cell population to uninfectedcontrol cells shows that the silenced cells display a very lowlevel of fluorescence, suggesting that there are only minimallevels of continuing transcription. Very few of the silenced cellshave lost the provirus, since most of them can be efficientlyreactivated by stimulation of the TCR (see Fig. 8B). Impor-tantly, the quiescent T cells can be maintained on the H80feeder cells for more than 6 months without any noticeable lossof viability or reactivation capability.

The progressive silencing of HIV gene expression as cellsenter quiescence can also be observed with viruses carryingwild-type Tat (Fig. 3A). In this experiment, 86.15% of the cellscarrying proviruses became silenced by day 63. After stimula-tion of the TCR by antibodies to CD3 and CD28, 90.99% ofthe cells were reactivated. As previously observed in our stud-ies with Jurkat T cells (40), the silencing of proviruses carryingthe wild-type Tat is generally slower and less efficient than thesilencing of proviruses carrying attenuated Tat genes.

The silencing of HIV proviruses was also observed in CD4�

T cells derived from tonsil tissues infected with viruses carryingeither H13L Tat or wild-type Tat and the mCherry fluorescentreporter (Fig. 3B). After 30 days, 52.35% of cells infected withviruses carrying the H13L Tat mutation and 46.79% of cellscarrying wild-type Tat were silenced. Because cells derivedfrom tonsil tissues show variable degrees of activation and areslower to enter quiescence than CD4� T cells isolated fromPBMC, the remaining experiments in the present study wereperformed with PBMC.

Latently infected cells have resting central memory cell phe-notype. In HIV patients undergoing HAART, the vast majorityof HIV-infected cells (over 89%) in the peripheral circulationare resting memory CD4� T cells, although a small but signif-icant fraction of infected cells (3%) have a naive CD4� T-cellphenotype (2). To document the changes that occur within thepopulation during the expansion and subsequent establishmentof the latent virus, the cells were stained with multiple anti-bodies to cell surface markers and examined using multicolorFACS. The most striking feature of this analysis is that theexpansion and culturing on H80 cells results in a more homo-geneous population than the CD4� cells originally isolatedfrom PBMC (Fig. 4).

As shown in Fig. 4A, freshly isolated T cells contain a mix-ture of naive T cells (40.43%, CD45RA� CD45RO), memorycells (24.61%, CD45RO� CD45RA), and a small population(19.01%) of dual-positive cells which represent cells that re-main in the transitional phase between naive and memory Tcells. In contrast, latently infected cells that have been culturedon H80 cells for 6 weeks display a uniform CD45RA

CD45RO� phenotype (92.98%), indicating that they are pri-marily resting memory cells (Fig. 4A and Fig. 5B).

To further define the phenotypes of the latently infected cells,we performed multicolor flow cytometric analysis (FACS) usingantibodies for a wide range of different cell surface protein mark-ers (Fig. 4, 5, and 6). The cells shown in Fig. 4 and 5 were infectedwith viruses carrying the H13L Tat gene.

As shown in Fig. 5A, 70.63% of the latently infected cellsused in these experiments did not express the d2EGFP markerand the remaining 28.77% expressed only low levels ofd2EGFP. However, 82.34% of the cells resumed HIV tran-scription and expressed high levels of d2EGFP after stimula-tion of the TCR.

CD38 is only expressed at very low levels on naive T cells andunactivated memory T cells, but is present at high levels on bothactivated T cells and mature thymocytes (11). Consistent withthese observations, we observed that freshly isolated naive andmemory cells express only low levels of CD38 (Fig. 4C). In con-trast, the latently infected memory (CD45RA CD45RO�) pos-itive cells showed uniformly high levels of CD38 (Fig. 4C, andhorizontal axis, Fig. 5A to F).

FIG. 1. Method for obtaining large populations of latently infectedprimary CD4� T cells.

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Activation of the cells through the TCR also resulted in theupregulation of the IL-2 receptor (CD25). In the experimentalresults shown in Fig. 4B, 96.32% of the cells upregulatedCD25, whereas 82.91% of the cells were activated in the ex-perimental results shown in Fig. 5C.

CD27 (Fig. 4E and 5D) is an important T-cell activation andexpansion marker, which is constitutively expressed on centralmemory T cells and strongly upregulated after TCR activationof naive cells (10). Upon acquisition of effector functions, the

levels of CD27 on central memory cells declines as CD27becomes soluble and detaches from the cell surface (18). CD27has also been linked with T-cell survival during expansion (21).In the latently infected population of cells, there is a low levelof CD27 on the cells surface that is modestly reduced afterTCR activation (Fig. 4E and 5D). This is consistent with theidentification of the latently infected cells as resting centralmemory cells.

The CCR7 receptors are the hallmark of activated central

FIG. 2. Progressive silencing of HIV expression in infected CD4� T cells. (A) Structure of lentiviral vectors. In some experiments, mCherrywas used in place of the d2EGFP fluorescent reporter depicted in this diagram. (B) Flow cytometric analysis of infected cells placed on H80 feedercells. The top panels show a light scatter analysis of the cell size. During proviral silencing, the activated T cells become quiescent and becomereduced in size, as indicated by reductions in both forward and side light scatter. The middle panels display selected histograms showing the fractionof cells expressing d2EGFP at day 0, day 20, and day 49 after cultivation of sorted cells on the H80 feeder cell line. A gray histogram showsuninfected control cells used to set the gates indicated by the bars. The percentage of cells in each gate for the various silenced cell populationsis given above the bars. The bottom panels show the time course of proviral silencing during cultivation of CD4� T cells on H80 feeder cells.Histograms are shown on the left. The graph on the right shows the percent d2EGFP� cells (green line; �2 � 100) and the percent d2EGFP

cells (black line; �2 � 100) for each time point (C).



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memory cells since CCR7 enhances the retention of these cellsby lymph nodes (46). In contrast, effector memory cells do notexpress CCR7 but do express other receptors that stimulatemigration to inflamed tissues (46). As shown in Fig. 4D and 5E,CCR7 is upregulated on the latently infected cells, which isagain consistent with their identification as a resting centralmemory cell population.

Finally, as d2EGFP levels increase (Fig. 5A), there is aconcomitant decrease in levels of CD4 (Fig. 5F). The down-regulation of CD4 is probably due to the increased expressionof HIV proteins (Vpu and Env) from the latent proviruses.

Very similar patterns of surface marker expression wereobserved in cells obtained from a second donor that wereinfected with viruses carrying the wild-type Tat gene (Fig. 6).In this cell population, 96.38% of the infected cells did notexpress the d2EGFP marker, and the remainder only ex-pressed low levels of d2EGFP. However, 66.45% of the cellsbecame strongly activated after stimulation of the TCR (Fig.6A). As in the previous experiment, the latently infectedcells are all CD45RA CD45RO� (Fig. 6B) and expressedlow levels of CD27 (Fig. 6D) and CCR7 (Fig. 6E), both ofwhich became somewhat upregulated after TCR activation.The cells also showed strong upregulation of CD25 (Fig. 6C)and strong downregulation of CD4 upon stimulation of theTCR (Fig. 6F).

Latently infected cells show reduced DNA synthesis and cellproliferation. To analyze the proliferation capability of thequiescent cells, we compared the incorporation of BrdU intocellular DNA before and after activating them with anti-CD3/CD28 antibodies (Fig. 7A) (29). For control cells we utilizedfreshly isolated, but not previously activated, CD4� T cellsisolated from PBMC. In parallel, we also performed intracel-lular staining for the nuclear antigen Ki67 (Fig. 7B) and CCR7(Fig. 7C). The IL-2 receptor chain, CD25, was included as anadditional dimension for the flow cytometry. Mock-infectedcells were used for these experiments to avoid interference ofthe d2EGFP signal expressed by the lentiviral vectors with thefluorescein isothiocyanate (FITC)-labeled antibodies utilizedin the BrdU incorporation assay.

As shown in Fig. 7, during cultivation on the H80 feedercells, the majority of the HIV-infected cells show stronglyreduced CD25 expression. In order to look for cells in thepopulation that were undergoing low levels of DNA synthesis,we labeled the cells for 18 h with BrdU, an unusually longlabeling period compared to the more typical 2-h “pulse” thatis used in cell cycle studies. Even under these conditions, in thelatently infected cell population, 32.55% of the cells failed toincorporate any BrdU (Fig. 7A). The remaining cells showedmeasurable levels of BrdU incorporation. As expected, afterTCR activation 96.88% of the latently infected cells incorpo-

FIG. 3. Epigenetic silencing of HIV expression CD4� T cells obtained from PBMC and tonsil tissue. (A) Silencing and reactivation in CD4�

T cells from PBMC. (Left panel) CD4� T cells isolated from PBMC were infected with viruses carrying wild-type Tat and the d2EGFP reporter.After sorting for d2EGFP� cells the population was cultivated on H80 feeder cells for up to 63 days. (Right panel) At day 63, the latently cellswere reactivated by stimulation of the TCR with �-CD3/CD28 antibodies. During the next 5 days there was a gradual reactivation of the entirelatently infected cell population. (B) Silencing in CD4� T cells from tonsils. CD4� T cells were isolated from discard tonsils and infected withviruses carrying either H13L Tat (left) or wild-type Tat (right) and the mCherry reporter. After sorting for mCherry� cells, the populations werecultivated on H80 feeder cells for the next 30 days. During this period there was progressive silencing of the proviruses.



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FIG. 4. Latently infected cells are central resting memory cells. Flow cytometric analysis of CD4� PBMC and quiescent CD4� T cells was performed.(A) CD45RA versus CD45RO. Note that the PBMC population contains a mixture of CD45RA� CD45RO naive T cells and CD45RA CD45RO�

memory T cells. (B) CD25 versus CD45RO. (C) CD38 versus CD45RO. (D) CCR7 versus CD45RO. (E) CD27 versus CD45RO.

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rated BrdU. Furthermore, the level of BrdU incorporationincreased 10-fold (Fig. 7A). As shown in Fig. 7B, the latentlyinfected cell population also had moderate levels of Ki67 ex-pression compared to freshly isolated and unstimulated CD4�

T-cell controls. Activation of latently infected cells throughthe TCR resulted in the strong upregulation of both Ki67and CD25 in over 96% of the cells (Fig. 7B). Thus, thelatently infected cells appear to be replicating slowly, butthey can be readily reactivated. As previously noted, CCR7expression is also upregulated when these cells were acti-vated (Fig. 7C).

NF-�B is necessary but not sufficient to activate latentlyinfected primary CD4� T cells. Work by Zack and coworkershas shown that, in contrast to Jurkat cells, induction of NF-�Bis necessary but not sufficient to induce latent proviruses innaive primary T cells (3). Similarly, we have found that TNF-�is unable to induce latent proviruses in central memory T cells(Fig. 8B), although it is a potent inducer of proviral expressionin latently infected Jurkat T cells (Fig. 8A). In contrast, asdescribed above, activation of the TCR strongly activates HIVtranscription in both Jurkat T cells (Fig. 8A) and latentlyinfected primary CD4� T cells (Fig. 8B). To confirm thatNF-�B was activated in the primary cells by both TNF-� andactivation of the TCR, we performed Western blots to measure

the nuclear levels of the NF-�B p65 subunit. As shown in Fig.8C, nuclear levels of p65 increase �10-fold after exposure ofthe quiescent cells to either activator.

We next examined whether P-TEFb levels are limiting inthese cells, as was originally reported by Rice and coworkersfor freshly isolated PMBC (22). As shown in Fig. 8C, nuclearextracts of the latent cells show very low levels of CDK-9 andCycT1. There is no increase in the nuclear levels of CDK-9 andCycT1 after exposure to TNF-�. In contrast, activation of theTCR leads to a �5-fold increase in CDK-9 and a �50-foldincrease in CycT1. This strongly suggests that P-TEFb levelsare limiting in the latently infected memory cells and that bothNF-�B and P-TEFb have to be activated in order to induceproviral transcription at both initiation and elongation steps oftranscription.

Latent proviruses in primary CD4� cells have heterochro-matic structures. Extensive studies in transformed cell sys-tems have shown that latent HIV proviruses typically con-tain high levels of histone deacetylases and heterochromaticstructures (9, 14, 35, 49, 50). It is believed that these chro-matin modifications are used to restrict transcription initi-ation and thus promote viral entry into latency (49). Inprimary cells, entry into latency is associated with differen-tiation events, leading to entry of cells into a quiescent state

FIG. 5. Changes in surface marker expression during the activation of cells latently infected with viruses carrying H13L Tat. Primary CD4� cellsharboring latent provirus which had been maintained on the H80 feeder cells were analyzed by multicolor flow cytometry before and afteractivation for 18 h with �-CD3/CD28 antibodies. (A) d2EGFP (GFP) versus CD38 (PerCP-Cy5). (B) CD45RA (APC) versus CD38 (PerCP-Cy5).(C) CD25 (PE) versus CD38 (PerCP-Cy5). (D) CD27 (APC-Cy7) versus CD38 (PerCP-Cy5). (E) CCR7 (PE-Cy7) versus CD38 (PerCP-Cy5)(F) CD4 (Pacific Blue) versus CD38 (PerCP-Cy5). Note that CD4 is downregulated while d2EGFP, CD25, and CCR7 are upregulated afterproviral activation.



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(31). Because it is not known whether some, or all, of thechromatin silencing mechanisms seen in the transformedcells are also observed in primary cells, we used ChIP assaysto study the chromatin of latent HIV proviruses in ourprimary CD4� T-cell model.

As shown in Fig. 9B, the latently infected CD4� T cells havelow, but detectable, levels of RNAP II at the promoter regionof the LTR (116 to �4). Similarly to Jurkat T cells, HDAC-1is also present at high levels, whereas the levels of acetylatedhistone H3 were very low. We have previously reported thatCBF-1 acts as an effective recruiter of HDACs to the HIVLTR (49). As shown in Fig. 9B, CBF-1 and its cofactors CIRand mSin3A are all present on the latent proviruses in primarycells. Induction of the latently infected cells by treating themwith �-CD3/CD28 antibodies increased RNAP II levels at thepromoter by �7-fold. In contrast, the levels of the repressorsCBF-1, CIR, mSIN3A, and HDAC-1 decreased substantially.As expected, treatment of latent primary CD4� lymphocyteswith �-CD3/CD28 antibodies resulted in NF-�B p65 and his-tone acetyltransferase p300 recruitment to the promoter. AfterTCR activation there was a 4- to 7-fold increase in acetylatedhistone H3 present at the provirus. The latent proviruses alsocarry heterochromatic markers at the promoter region of the

LTR, including trimethylated histone H3 at positions K9 andK27 (Fig. 9B). HP1-�, a heterochromatic protein that bindsexclusively to trimethyl-K9-H3 histones, is also present at theHIV LTR (14). After TCR activation, there were significantdecreases in the levels of trimethylated histone H3 (K9 andK27) and in the HP1-� repressor protein, whereas the levels ofacetylated histones rise at the HIV LTR.

Similar results were obtained by using primers directed tothe upstream nucleosome 0 region of the LTR (Fig. 9A) andthe downstream nucleosome 1 (Fig. 9C) and nucleosome 2(Fig. 9D) regions. Because the resolution of the ChIP assay isbetween 500 and 700 bp, there is some signal overlap betweeneach of these adjacent regions. Nonetheless, some importantdifferences in protein distribution can be observed. The mostnotable difference is that there are much higher levels of his-tones present in the nucleosome 0 and 1 regions than at thepromoter of the latent proviruses. These observations are con-sistent with previous studies with transformed cell lines thatdemonstrated that the HIV promoter region is relatively de-void of histones (51). The data also demonstrate that modifiedhistones are present both upstream and downstream of thetranscription start site. In contrast, as expected, p65 is largelyrestricted to the promoter region (Fig. 9D).

FIG. 6. Changes in surface marker expression during the activation of cells latently infected with viruses carrying wild-type Tat. Primary CD4�

cells infected with viruses carrying wild-type Tat were allowed to enter latency by cultivation on H80 feeder cells for over 60 days. Cells harboringlatent provirus were analyzed by multicolor flow cytometry before and after activation for 16 h with �-CD3/CD28 antibodies. (A) d2EGFP (GFP)versus CD45RO (PE). (B) CD45RA (APC) versus CD45RO (PE). (C) CD25 (Perp-Cy5) versus CD45RO (PE). (D) CD27 (APC-Cy7) versusCD45RO (PE). (E) CCR7 (PE-Cy7) versus CD45RO (PE). (F) CD4 (Pacific Blue) versus CD45RO (PE). Note that the latently infected cellsconstitutively express CD45RO, CD38, and CD27. The cells used in this experiment were from a different donor than the cells used in theexperiment in Fig. 5.



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DISCUSSION

The recognition that an extremely stable HAART-resistantreservoir of latent HIV is present in most patients has focusedattention on the molecular mechanisms governing HIV la-tency. Unfortunately, both because it is difficult to isolate largenumbers of latently infected cells from patient samples andbecause there have been problems in developing culture con-ditions that allow study of HIV latency in primary cells, virtu-ally all molecular investigations of HIV latency have involvedthe use of transformed cell lines, such as Jurkat T cells. Thesestudies have suggested that there are many close parallels inthe behavior of the latent proviruses in transformed cells andprimary cells. For example, in both cell types, latent provirusesare preferentially integrated into actively transcribed genes

(19, 34) and the addition of HDAC inhibitors induces latentproviruses, suggesting that chromatin restrictions play a uni-versal role in maintaining latency (54). However, because thequiescent phenotype of the infected CD4� T cells that makeup the bulk of the latent reservoir is drastically different fromthe constitutively activated Jurkat T cells, important differ-ences in the viral reactivation and shutdown pathways are alsolikely to exist. Thus, there was a pressing need to developreliable and biochemically tractable model systems to studyHIV latency in primary CD4� T cells.

Recent reports have demonstrated that it is possible to gen-erate latent HIV infection in primary human CD4� T cellsusing in vitro maturation (5), coculture (45), spinoculation(48), or manipulation of the interleukin levels (26, 36). Al-

FIG. 7. CD4� cells show restricted DNA synthesis after cultivation on H80 feeder cell lines. (A) The proliferation capability of freshly isolatedCD4� T cells from PBMC and mock-infected cells that had been cultivated on the H80 feeder cell line for more than 60 days was analyzed bymeasuring BrdU nucleotide incorporation into cellular DNA (FITC) before and after activating them with �-CD3/CD28 antibodies. Labeling withBrdU was done for 18 h. The data are displayed against a second cell activation marker, CD25 (APC-Cy7). (B) Ki67 (PE) expression afteractivation of quiescent T cells. (C) CCR7 expression after activation of the quiescent T cells.



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though these are promising developments, none of these meth-ods yield sufficiently large quantities of latently infected cells toperform extensive biochemical analyses.

A frequently encountered problem in attempts to generatelarge homogeneous populations of latently infected primary Tcells is that infected T cells rapidly enter apoptosis once TCRsignaling is interrupted. This leads to massive cell loss during invitro T-cell differentiation by specific cytokine exposure re-gimes (26, 36). Consequently, the recent ex vivo systems used togenerate latently infected primary cells typically only yield lessthan 20% viable cells at the end of resting phase. Furthermore,in both the method of Bosque and coworkers (26) and themethod of Marini (36) a low frequency of HIV infection resultsin less than 10% of the recovered cell populations carryingproviruses. The Siliciano laboratory has been exploring a po-tential solution to this problem through the transduction ofBcl-2, an antiapoptotic factor that allows the primary CD4� Tcells to survive in a quiescent state in vitro (53). Althoughpromising, this strategy raises the concern that overexpressionof Bcl-2 may alter the normal physiology of primary restingCD4� T cells. Similarly, the use of IL-7 in the method ofMarini et al. (36) raises concerns that these cells may remainpartially activated, since IL-7 is known to be a potent inducerof latent HIV (47).

We have described here a novel primary CD4� T-cell-basedmodel system that allows us to obtain large populations ofpure, latently infected, cells. The model is based on our pre-vious observation that lentiviral vectors carrying an attenuatedTat mutation (H13L) rapidly and efficiently enter latency. Inadopting this strategy for use with primary cells, a key step wasto utilize the feeder layer system established by Sahu et al. (45)to maintain infected CD4� T cells in the absence of TCRstimulation.

Our method has proven to be highly reproducible and robustduring the last 2 years. During the course of this work, we haveproduced latently infected cells from six different donors usingfour different viral vectors carrying either wild-type or H13LTat and either the d2EGFP or mCherry fluorescent reporters.In general, we have found that it was useful to include theattenuated H13L Tat mutant in our experiments, since virusescarrying attenuated Tat enter latency more efficiently thanviruses carrying the wild-type Tat. It is therefore not surprisingthat recent viral isolation studies have shown that latentlyinfected cells obtained from patients tend to accumulate Tatvariants with reduced transactivation potential (55).

Extensive multicolor flow cytometric analysis of the latentlyinfected T cells shows that they represent a very homogeneouspopulation of cells that is CD45RA, CD45RO�, CD38�,CD25, CD27�, and CCR7�. This combination of surfacemarkers suggests that these cells represent a central memoryT-cell population (39). The cells are largely, but not com-pletely, quiescent since although they do not divide, they arestill able to incorporate BrdU and express moderate levels ofKi67. This partially activated phenotype may be a consequenceof the IL-2 present in the culture medium, which is needed tomaintain cell viability.

A striking result from these studies is that, just as we havepreviously observed in Jurkat T cells, there is progressive epi-genetic shutdown of HIV transcription as the virus enters la-tency. Using ChIP assays we have shown that latent proviruses

FIG. 8. Latently infected primary CD4� T cells have restrictednuclear P-TEFb levels. (A) Latently infected Jurkat T cells (clone2D10 [40]) were stimulated for 18 h by TNF-� (left) or by �-CD3/CD28 antibodies (right) and analyzed for d2EGFP expression by flowcytometry. The gray histogram shows uninfected control cells used toset the gates, indicated by the bars above the histograms. The percent-age of cells in each gate for the activated cell population is given abovethe bars, and the percentage of cells in each gate for the controlunstimulated cell population is given below the bar. (B) CD4� T cellsfrom PBMC were infected with pHR-p-d2EGFP vector carrying H13LTat and allowed to enter latency by culturing on H80 feeder cells. Thelatently infected cell population was then stimulated for 18 h by TNF-�(left) or by �-CD3/CD28 antibodies (right) and analyzed for d2EGFPexpression by flow cytometry. The gray histogram shows uninfectedcontrol cells used to set the gates, indicated by the bars above thehistograms. The percentage of cells in each gate for the activated cellpopulation is given above the bars, and the percentage of cells in eachgate for the control unstimulated cell population is given below thebar. (C) Western blotting was performed on nuclear extracts obtainedfrom latently infected cells before and after activating them with eitherTNF-� or with antibodies against TCR. Antibodies used were:�-NF�B p65, �-CDK9, �-CyclinT1 (CycT1), and �-SPT5. Note thatNF-�B p65 is efficiently induced by the both TNF-� and TCR stimu-lation. In contrast, P-TEFb levels (CDK-9, CycT1) in the nucleus arestrongly stimulated by TCR activation but not by TNF-� treatment.



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recruit HDACs via CBF-1 and its cofactors CIR and mSIN3A.We were also able to demonstrate that the latent HIV LTRcontains stable heterochromatic structures. Histone H3 tri-methylated at positions K9 and K27 and the HP-1� protein areall present. These results are consistent with the hypothesisthat establishment of restrictive heterochromatic structures onthe latent HIV provirus involves a series of sequential eventsstarting with the recruitment of HDACs to the promoter viaCBF-1, followed by methylation of histones by Suv39H1 andbinding of the HP proteins to the methylated histones (14, 35,49). Since the ChIP experiments were performed with mixedpopulations of cells that contained thousands of separate in-tegration events, rather than clones, we can conclude that theepigenetic silencing we observed is due to recognition of spe-cific features of the HIV proviral genome rather than a con-sequence of integration into a specific site.

As in the Jurkat T-cell models, activation of latently infectedprimary CD4� lymphocyte by stimulation of the T-cell recep-tor using �-CD3/CD28 antibodies reverses these chromatin

modifications. After TCR activation, there are significant re-ductions in the levels of trimethylated histone H3 (K9 andK27) and a concomitant fall in the levels of HP1 repressorprotein. TCR activation also resulted in the reversal of theacetylation status of histones represented by the higher pres-ence of acetylated histone 3 at the LTR. However, the reacti-vation of latent proviruses in primary cells is more complexthan proviral reactivation in Jurkat T cells. Most importantly,although TNF-� is an extremely potent inducer of latent pro-viruses in Jurkat T cells, it is totally ineffective in primary cells.The failure of TNF-� to activate latent proviruses in primarycells appears to be due to limiting quantities of free P-TEFb,which we have measured by examining the levels of CDK-9 andCycT1 present in nuclear extracts. Under the extraction con-ditions we have used, the large, inactive form of P-TEFb isfound in the cytoplasmic fraction (1). Consistent with obser-vations made in freshly isolated PMBCs (22), activation of thecells through the TCR produces a dramatic upregulation ofP-TEFb. In our experiments, this activation of P-TEFb is dem-

FIG. 9. Fluctuations in the levels of different transcription and chromatin-associated factors, before and after activation of latently infectedprimary CD4� T cells with �-CD3/CD28 antibodies. ChIP analysis was performed on primary cells latently infected with proviruses carrying H13LTat and the d2EGFP marker. Antibodies used for the analysis included RNAP II (N20), transcription initiation factors (p65 and p300), CBF-1repressor complex (CBF-1, CIR, and Sin3A), and histones and chromatin-modifying proteins (HDAC-1, acetylated histone H3, trimethyl-lysine-9-histone H3, trimethyl-lysine-27-histone H3, and HP-1�). (A) Primers directed to the nucleosome 0 region (396 to 282). (B) Promoter region(116 to �4). (C) Nucleosome 1 (�30 to �134). (D) Nucleosome 2 (�286 to �390). Blue bars, latently infected cells; red bars, cells after 30 minof treatment with �-CD3/CD28 antibodies.



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onstrated by the �20-fold increase in the levels of CDK-9 andCycT1 in the nuclear extracts after activation of latently in-fected cells by antibodies to CD3 and CD28.

In summary, the model system we have described presentsexciting opportunities to study specific molecular aspects ofboth the entry and exit from HIV latency. Using our method,latently infected cells carrying informative mutations in theviral LTR and regulatory genes can be readily produced. Fur-thermore, since we have been able to generate pure popula-tions of latently infected primary T cells in amounts suitablefor biochemical studies, our model should facilitate the screen-ing of drug candidates as part of the effort to develop newtherapeutic approaches designed to eliminate latent HIV res-ervoirs.

ACKNOWLEDGMENTS

We thank our coworkers Scott Sieg and Douglas A. Bazdar forassistance in the multicolor flow cytometry, and we thank laboratorymembers Joseph Hokello, Uri Mbonye, Julian Wong, Julia Friedman,Julie Jadlowsky, Amzie Pavlisin, Kara Lassen, and Zafeiria Athanasioufor their help and fruitful discussions. Darell Bigner (Duke University)and Miles Cloyd (UT Galveston) kindly provided the H80 cells. Wealso thank the CWRU/UH Center for AIDS Research (P30-AI036219)for the provision of flow cytometry services.

This study was supported by NIH grants R01-AI067093 and DP1-DA028869 to J.K.

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Establishment of HIV Latency in Primary CD4 Cells Is due to